Generation of Functional Immortalized Human Corneal Stromal Stem Cells.

In addition to their therapeutic potential in regenerative medicine, human corneal stromal stem cells (CSSCs) could serve as a powerful tool for drug discovery and development. Variations from different donors, their isolation method, and their limited life span in culture hinder the utility of primary human CSSCs. To address these limitations, this study aims to establish and characterize immortalized CSSC lines (imCSSC) generated from primary human CSSCs. Primary CSSCs (pCSSC), isolated from human adult corneoscleral tissue, were transduced with ectopic expression of hTERT, c-MYC, or the large T antigen of the Simian virus 40 (SV40T) to generate imCSSC. Cellular morphology, proliferation capacity, and expression of CSSCs specific surface markers were investigated in all cell lines, including TNFAIP6 gene expression levels in vitro, a known biomarker of in vivo anti-inflammatory efficacy. SV40T-overexpressing imCSSC successfully extended the lifespan of pCSSC while retaining a similar morphology, proliferative capacity, multilineage differentiation potential, and anti-inflammatory properties. The current study serves as a proof-of-concept that immortalization of CSSCs could enable a large-scale source of CSSC for use in regenerative medicine.

Long-Term Efficacy and Safety of Subconjunctival/Perilesional 5-Fluorouracil Injections for Ocular Surface Squamous Neoplasia.

PURPOSE: To investigate the effectiveness and safety of subconjunctival/perilesional 5-fluorouracil injections on ocular surface squamous neoplasia (OSSN) during a 3-year follow-up period. PATIENTS AND METHODS: We followed up six patients with intraepithelial OSSN (in one eye each) that had regressed after subconjunctival/perilesional 5-fluorouracil injections. Conjunctival fluorescein angiography (FA) and indocyanine green angiography (ICGA), as well as anterior segment optical coherence tomography (AS-OCT), were performed to evaluate the OSSN status 3 years after initiation of treatment. RESULTS: The mean age of patients (five males, one female) at baseline was 62.3±11.6 years. The mean number of 5-fluorouracil injections was 17.0±8.6, with a mean treatment duration of 13.0±7.4 weeks. At the final visit, both intratumoral and conjunctival feeding vessels had disappeared on ICGA and FA, with no neovascularization-related leakage, in accordance with the results of AS-OCT. The period from complete tumor regression to final visit according to AS-OCT was 32.5±4.2 months, which was longer than that according to ICGA (31.3±3.2 months, p=0.034). The final best-corrected visual acuity was similar to that at baseline (p=0.128). No side effects were observed in any of the eyes. CONCLUSION: Subconjunctival/perilesional 5-fluorouracil injections are an effective and safe treatment for OSSN. Future studies with a larger sample size are warranted for confirmation of our findings, as well as investigation into the reasons for residual areas of non-perfusion in the conjunctiva.

Limbal Stem Cell Deficiency After Glaucoma Surgery.

PURPOSE: To characterize the clinical presentation of limbal stem cell deficiency (LSCD) associated with glaucoma surgeries. METHODS: This is a retrospective cross-sectional study of patients with LSCD and glaucoma who presented to the Stein Eye Institute at the University of California, Los Angeles, between 2009 and 2018. Patients who underwent trabeculectomy and/or aqueous shunt surgery were included. The severity of LSCD was staged using global consensus guidelines and a clinical scoring system, and basal epithelial cell density was measured by in vivo confocal microscopy. Anatomic locations of glaucoma and non-glaucoma surgeries, locations of LSCD, and severity of LSCD were compared. RESULTS: Fifty-one eyes of 41 patients with LSCD associated with glaucoma surgery were included in this study. LSCD in these patients uniquely featured sectoral replacement of corneal epithelium by conjunctival epithelium, without corneal neovascularization or pannus. The sites of glaucoma surgery strongly correlated with the locations of LSCD (P = 0.002). There was a trend toward increased severity of LSCD in eyes with 2 or more glaucoma surgeries as compared to eyes with 1 glaucoma surgery, although the difference did not reach statistical significance (P = 0.3). Use of topical glaucoma medications correlated with LSCD severity, while the impact of antimetabolites did not reach statistical significance. The location of glaucoma drainage surgery is correlated with the location of LSCD. CONCLUSIONS: LSCD associated with glaucoma surgery has clinical features distinct from LSCD resulting from other etiologies. Further study is required to delineate the full impact of glaucoma surgery on limbal stem cell function and survival.

Evaluation of Cryopreservation Media for the Preservation of Human Corneal Stromal Stem Cells.

Introduction: Human corneal stromal stem cells (CSSCs) have gained increasing attention in the treatment of corneal stromal scars. In view of this, the preparation and storage of CSSCs are critical to maintaining the regenerative potential of CSSCs. The goal of the study was to investigate the human serum (HS) concentration in the cryomedia that could best preserve CSSCs. Materials and Methods: Three different cryopreservation media, varying in HS concentration were evaluated in their ability to preserve the viability and phenotype of CSSCs: 2% HS (FS1), 4% HS (FS2), and 90% HS (FS3). After thawing, CSSCs morphology, recovery rate, cell proliferation, relative gene expression of CSSC markers (ABCG2, SOX2, NANOG, PAX6, and SIX3), and their anti-inflammatory response (level of TNFAIP6) were compared with those of unfrozen CSSCs (control). Results: Cryopreserved CSSCs had similar cell morphology as the control. Cell viability was significantly higher using FS2 (92.7 ± 1.3%) compared with FS1 (88 ± 0.8%, p = 0.018). Doubling times of CSSCs were maintained in all cryopreserved conditions, as in the control (p > 0.05), which were 0.9 ± 0.1 days and 1.8 ± 0.0 days at passages 3 and 4, then increased to 18.2 ± 1.9 days at passage 6 (p > 0.05). The expression level of stem cell/progenitor cell markers investigated was not affected by the cryopreservation with any of the three media. In addition, cryopreserved CSSCs have a similar expression level of TNFAIP6 after stimulation with proinflammatory cytokines as the control (p > 0.05). Conclusion: Our results indicated that all three cryopreservation media maintained CSSCs phenotype after undergoing one freezing/thawing cycle. Impact Statement Corneal stromal stem cells (CSSCs) offer an alternative for the treatment of corneal stromal scars. Cryopreservation of CSSCs is necessary as it enables feasibility of using CSSCs as a cell therapy candidate. The current study shows that media used to cryopreserve CSSCs could be optimized to maintain cell viability, phenotype, and potency of CSSCs after thawing.

Ocular surface squamous neoplasia: angiographic characteristics and response to subconjunctival/perilesional 5-fluorouracil injections.

Introduction: To investigate the angiographic characteristics of ocular surface squamous neoplasia (OSSN) and to evaluate the efficacy of subconjunctival/perilesional 5-fluorouracil injections in OSSN cases. Materials and methods: Six eyes of six patients with primary OSSN, received perilesional, subconjunctival, 25-mg/mL 5-fluorouracil injections at certain intervals. Anterior segment digital photography images, anterior segment optical coherence tomography (AS-OCT), and conjunctival indocyanine green angiography (ICGA) were obtained simultaneously with fluorescein angiography. Results: The mean best-corrected vision acuity significantly improved after treatment. At baseline, the median of the largest thickness of OSSN was 905.0 (interquartile range: 492.0-1592.5) µm based on AS-OCT data. There was an abrupt transition between normal and abnormal epithelium, a thickened hyper-reflective epithelium, and a sharp plane of cleavage between the lesion and underlying tissue, all indicative of OSSN. The angiographic characteristics of OSSN included focal or seafan-shaped intratumoral and conjunctival feeding vessels visible via ICGA, and abnormal vascular leakage visible with fluorescein angiography. The median time to tumor regression after treatment was 35.0 (interquartile range: 32.0-45.5) days in five eyes without recurrence, and OSSN in one eye regressed partially 40 days after treatment. Conclusion: This is the first report of the angiographic characteristics of OSSN and its response to subconjunctival/perilesional 5-fluorouracil injections by simultaneous conjunctival angiography and AS-OCT. The improved subconjunctival/perilesional 5-fluorouracil injection was an effective therapy for OSSN in both best-corrected vision acuity gain and anatomic outcomes.

Mertk gene expression and photoreceptor outer segment phagocytosis by cultured rat bone marrow mesenchymal stem cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSCs) are multipotential stem cells that have been used for a broad spectrum of indications. Several investigations have used BM-MSCs to promote photoreceptor survival and suggested that BM-MSCs are a potential source of cell replacement therapy for some forms of retinal degeneration. PURPOSE: To investigate the expression of the MER proto-oncogene, tyrosine kinase (Mertk), involved in the disruption of RPE phagocytosis and the onset of autosomal recessive retinitis pigmentosa in rat BM-MSCs and to compare phagocytosis of the photoreceptor outer segment (POS) by BM-MSCs and RPE cells in vitro. METHODS: MSCs were isolated from the bone marrow of Brown Norway rats. Reverse transcription-PCR (RT-PCR) and western blot analyses were used to examine the expression of Mertk. The phagocytized POS was detected with double fluorescent labeling, transmission electron microscopy, and scanning electron microscopy. RESULTS: Mertk expression did not differ among the first three passages of BM-MSCs. Mertk gene expression was greater in the BM-MSCs than the RPE cells. Mertk protein expression in the BM-MSCs was similar to that in the RPE cells in the primary passage and was greater than that in the RPE cells in the other two passages. BM-MSCs at the first three passages phagocytized the POS more strongly than the RPE cells. The process of BM-MSC phagocytosis was similar to that of the RPE cells. CONCLUSIONS: BM-MSCs may be an effective cell source for treating retinal degeneration in terms of phagocytosis of the POS.

Curative effect assessment of bandage contact lens in neurogenic keratitis.

AIM: To observe the curative effect of bandage contact lens in neurogenic keratitis. METHODS: Twenty cases of neurogenic keratitis were studied at the Department of Ophthalmology, the first Affiliated Hospital of China Medical University, between October 2012 and June 2013. These included 13 males and 7 females, aged from 35 to 88y. Patients were voluntarily divided into an experimental group (lens wearing group, n=10) and control group (drug therapy, n=10). In experimental group patients wore silicone hydrogel bandage soft contact lens. Both groups used the following eyedrops: 0.5% levofloxacin TID; 0.5% Sodium carboxymethyl cellulose QID; fibroblast growth factor BID; ganciclovir BID [cases complicated with herpes simplex virus (HSV)]; compound tropicamide BID (cases concurrent hypopyon). The healing time of corneal ulcer and complication rates were observed in the two groups. RESULTS: The healing time of corneal ulcer in the experimental group was 10.80±4.44d versus 46.70±13.88d in the control group (P<0.05). No complications occurred in the experimental group, except for the lens falling off twice in one case, the patient recovered eight days after rewearing the lens. While in the control group, all cases vascularized, 2 cases were complicated with descemetocele that recovered with amniotic membrane transplantation and 1 case was complicated with corneal perforation that recovered by autologous conjunctival flap covering. CONCLUSION: Bandage contact lens is a safe and effective method of treating neurogenic keratitis and significantly shortened the healing time of corneal ulcer.

Pathological characteristics of the different stages of Acanthamoeba keratitis.

AIMS: To classify the clinical stages of Acanthamoeba keratitis (AK), and clarify the relationship between pathological changes and clinical features. METHODS AND RESULTS: Between January 2007 and May 2012, AK was diagnosed in 11 eyes by pathological examination and confocal laser scanning microscopy. Pathological investigation of all cornea samples from keratoplasty was done with periodic acid-Schiff and haematoxylin and eosin stains. AK clinical stage, pathological features and postoperative treatment were studied retrospectively. The 11 cases were classified into development stage, convalescence stage, or cicatricial stage. In the development stage, marked conjunctival hyperaemia, a corneal epithelial defect, obvious corneal infiltration and progressive inflammation were seen; pathological changes comprised abundant inflammatory cells and a rounded cyst in the oedematous stroma, as well as a very small amount of neovascularization. In the convalescence cases, moderate conjunctival hyperaemia, corneal disciform structures, repair of the corneal epithelial defect and abundant neovascularization were seen; pathological changes included significant tissue necrosis and a small, shrunken cyst in the stroma, as well as significant neovascularization. In the cicatricial stage, keratoleukoma was seen; pathological changes comprised a few inflammatory cells and shrunken cysts scattered in the stroma. There were no cases of recurrence. CONCLUSIONS: The pathological features of different clinical stages confirmed the new clinical classification of AK.

[The relationship between MERTK gene and protein kinase C in the phagocytic process of human retinal pigment epithelial cells].

OBJECTIVE: To investigate relationship of the expression of MERTK gene and the activity of protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (hRPE) cells. METHODS: Cultured hRPE cells were incubated with rod outer segments (ROS) suspension (containing ROS 1x10(10)/L) at 37 degrees C, then cells were rinsed at different times to terminate the phagocytosis. The kinetic of phagocytosis was measured by double-fluorescent labeling. The activity of PKC and the expression level of MERTK gene were measured by counting gamma-32P radio-activity with liquid scintillation and RT-PCR respectively. Change of MERTK gene expression was measured after hRPE was treated cells with stimulator or inhibitor of PKC. Statistical analysis was performed by SPSS 13.0 software. RESULTS: The phagocytic assay showed that the quality of bound and ingested ROS by hRPE cells increased. The quality of ingested ROS by hRPE cells at 24 hours was (2.85+/-0.11)x10(6), which reached maximum contrast with control (0.00+/-0.00)x10(6) (t=47.64, P<0.05). The activity of PKC (both in cytoplasm and on membrane) decreased during all the incubation periods compared with control [cytoplasm: (329.63+/-14.26) nmolxg(-1)xmin(-1)and on membrane: (467.67+/-68.87) nmolxg(-1)xmin(-1)], and reached the minimum at 24 h[cytoplasm: (151.13+/-17.67) nmolxg(-1)xmin(-1) and on membrane: (152.45+/-64.83) nmolxg(-1)xmin(-1); cytoplasm t=89.66 and membrane t=10.31, P<0.05]. The level of MERTK mRNA increased in pulse-chase and long-time incubation test. The gray level for 90 min was 1.8853+/-0.0077, contrasted with control 0.7246+/-0.0062, F=16,060.2167 and P<0.05. The gray level for 24 h was 0.5946+/-0.0082, contrasted with control 0.3343+/-0.0064, F=919.8421 and P<0.05. When up-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA was decreased in the proceeding incubating with ROS contrasted with control (pulse-chase group F=17,142.2331, long time group F=1886.4614; P<0.05). After down-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA waved between 4.4670+/-0.0092 and 5.7034+/-0.0095 in the first 30 min of incubating with ROS, which lower than control 0.9117+/-0.0021 (F=199,012.9138, P<0.05). CONCLUSIONS: The lower activity of PKC and the higher expression MERTK gene are very important for sustaining phagocytic process of ROS by hRPE cells. MERTK gene and PKC both as up-stream regulators are negative-correlated in the phagocytic process of hRPE.

[The alteration of cyclic adenosine monophosphate and protein kinase C in the phagocytic process of human pigment epithelial cells].

OBJECTIVE: To investigate the role of cyclic adenosine monophosphate (cAMP) and protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (HRPE) cells by measuring phagocytosis indexes in specific and nonspecific phagocytic process. METHOD: The cultured HRPE cells were incubated with 1 x 10(7)/ml rod outer segments (ROS) or latex beads (LB) at 37 degrees C, then the phagocytosis were terminated at different incubation time (5 min-48 h). The kinetics of phagocytosis was measured by double-fluorescent vital assay. The binding and ingestion of ROS or LB were proved by scanning and transmission electron microscope. cAMP level was measured using (125)I-cAMP radio-immunity kit, and PKC activity was measured by counting gamma-(32)P radio-activity with liquid scintillation. RESULT: In specific phagocytosis of HRPE cells, the binding of ROS was happened at 15 min of incubation, and the cAMP level was decreased at the same time; the activity of PKC (both in cytoplasm and membrane)was decreased at 5 min which was earlier than the changes of the level of cAMP, but both indexes were reached their lowest level at 24 h. In nonspecific phagocytosis of HRPE cells, the binding of LB was happened at 1.5 h; on the other hand, when HRPE cells were incubated with LB, cAMP level did not change until 12 h (P > 0.05), but it began to decrease in the incubation time from 12 h-48 h; the activity of PKC (both in cytoplasm and on membrane) was stable during all incubation periods (P > 0.05). CONCLUSION: cAMP and PKC are very important for sustaining the ingestion process in specific phagocytosis of HRPE cells, but have no direct relationship with nonspecific phagocytosis. Furthermore, cAMP level and PKC activity in HRPE cells are decreased during specific phagocytosis.